JP2009254315A - Method for producing ester derivative - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an acrylate compound useful as a monomer for a polymer material by reacting a (meth)acrylic acid derivative with an aliphatic carboxylic acid or polybasic carboxylic acid which may have hydroxyl groups by using enzymatic reaction. <P>SOLUTION: Provided is a method for producing a (meth)acrylate by the esterification reaction of an aliphatic carboxylic acid or polybasic carboxylic acid which may have hydroxyl groups with a hydroxyacrylate under a mild condition using a lipase derived from a specific microorganism or an arming yeast presenting the lipase on the cell surface layer. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention is useful as a polymer material monomer characterized by reacting a (meth) acrylic acid derivative with an aliphatic carboxylic acid which may have a hydroxyl group or a polyvalent carboxylic acid using an enzymatic reaction. The present invention relates to a method for producing an acrylate compound.

A (meth) acrylic acid derivative having a hydroxyl group represented by 2-hydroxyethyl (meth) acrylate (HE (M) A) is used as a raw material for various acrylic resins. By introducing a hydroxyl group into an acrylic resin, it becomes possible to impart water solubility to the resin or a crosslinking reaction by reaction with a polyisocyanate compound, and is widely used in the fields of paints and adhesives / adhesives. However, when an acrylate such as HEMA is copolymerized with an acrylic resin, there is a problem that physical properties of the cured product with the polyisocyanate compound become brittle because a hydroxyl group exists in the vicinity of the main chain of the acrylic resin. In order to solve such a problem, a monomer having a (meth) acryl group introduced at one end, such as PEG, is used, but there is a problem such as an increase in water absorption.

On the other hand, development of materials using plant-derived materials as renewable resources has been actively conducted in recent years. As a highly hydrophobic (meth) acrylate compound using a fatty acid as a raw material and a production method thereof, there are disclosures of Patent Document 1, Patent Document 2, and Patent Document 3 below.
However, when a raw material unstable to heat, acid, oxygen or the like is used, there are problems such that it may not be possible to produce due to side reactions such as remarkable coloring, decomposition, and gelation.
Furthermore, in the esterification reaction using hydroxy fatty acid and alkanol acrylate as raw materials, in addition to the cross-reaction between the carboxyl groups of alkanol acrylate and hydroxy-fatty acid, the method in the above-mentioned patent document for the cross-reaction between each hydroxyl group However, there is a problem that a process such as functional group protection / deprotection of the hydroxyl group is further required.

JP 2-110115 A Japanese Patent Laid-Open No. 5-229991 Patent No. 3829101

The problem to be solved by the present invention is characterized by reacting a (meth) acrylic acid derivative, an aliphatic carboxylic acid which may have a hydroxyl group, or a polyvalent carboxylic acid using an enzymatic reaction. The present invention provides a method for producing an acrylate compound useful as a polymer material monomer.

As a result of extensive research, the present inventors have found that the genus Aspergillus oryzae, the genus Achromobacter, the genus Bacillus, the genus Candida, the genus Chromobacter, and Fusarium (Fusum). ) Genus, Humicola genus, Hyphozyma genus, Pseudomonas genus, Rhizomucor genus, Rhizopus genus, or Thermomyces cerevisiae (Thermoperis genus) Using arming yeast (B) (hereinafter referred to as lipase arming yeast (B)) presented on the cell surface. Under the conditions, an esterification reaction between a (meth) acrylic acid derivative and an aliphatic carboxylic acid having 6 to 24 carbon atoms which may have a hydroxyl group, found a method for producing an acrylate compound useful as a polymer material monomer The present invention has been completed.

That is, the present invention provides a compound represented by the general formula (1) in the presence of lipase (A) or lipase arming yeast (B).

(In the formula, R ₁ represents a hydrogen atom or a methyl group, X represents an oxygen atom or a nitrogen atom, and n represents an integer of 2 to 4.)
(Meth) acrylic acid derivatives represented by the general formula (2)

(In the formula, R ₂ represents a C 5-23 alkyl group or alkenyl group which may have a hydroxyl group or a carboxy group.)
General formula (3) for reacting with a carboxylic acid represented by

(In the formula, R ₁ is a hydrogen atom or a methyl group, X is an oxygen atom or a nitrogen atom, n is an integer of 2 to 4, and R ₂ is an alkyl group having 5 to 23 carbon atoms which may have a hydroxyl group or a carboxy group. Or represents an alkenyl group.)
It is related with the manufacturing method of the acrylate compound represented by these.

According to the present invention, an acrylate compound useful as a polymer material monomer characterized by reacting a (meth) acrylic acid derivative with an aliphatic carboxylic acid which may have a hydroxyl group or a polyvalent carboxylic acid. A manufacturing method can be provided.

In the presence of the lipase (A) or the lipase arming yeast (B), the present invention provides a compound represented by the general formula (1)

(In the formula, R ₁ represents a hydrogen atom or a methyl group, X represents an oxygen atom or a nitrogen atom, and n represents an integer of 2 to 4.)
(Meth) acrylic acid derivatives represented by the general formula (2)

(In the formula, R ₂ represents a C 5-23 alkyl group or alkenyl group which may have a hydroxyl group or a carboxy group.)
General formula (3) for reacting with a carboxylic acid represented by

(In the formula, R ₁ is a hydrogen atom or a methyl group, X is an oxygen atom or a nitrogen atom, n is an integer of 2 to 4, and R ₂ is an alkyl group having 5 to 23 carbon atoms which may have a hydroxyl group or a carboxy group. Or represents an alkenyl group.)
The manufacturing method of the acrylate compound represented by these is provided.

The compound represented by the general formula (1) can be obtained by a generally known method by a dehydration condensation reaction between (meth) acrylic acid and a dihydric alcohol or hydroxyamine.
In the formula, R ₁ may be a hydrogen atom or a methyl group. When X represents an oxygen atom, the compound represented by the general formula (1) is an ester derivative, and when X represents a nitrogen atom, the compound represented by the general formula (1) is an amide derivative. However, either may be used in the present invention. n can be used without particular limitation as long as it is an integer of 2 to 4, but it is particularly preferably n = 2.
In addition, the aliphatic carboxylic acid subjected to the ester reaction with the compound represented by the general formula (1) is represented by the general formula (2).

(In the formula, R ₂ represents a C 5-23 alkyl group or alkenyl group which may have a hydroxyl group or a carboxy group.)
The R ₂ may or may not have a hydroxyl group or a carboxy group depending on the use of the ester derivative obtained.
Examples of the carboxylic acid having 6 to 24 carbon atoms that may have a hydroxyl group used in the present invention include, for example, caproic acid, caprylic acid, capric acid, undecylenic acid, palmitic acid, oleic acid, stearic acid, and ricinol. Examples thereof include aliphatic carboxylic acids such as acid and hydroxystearic acid, but are not limited thereto.
Furthermore, as the carboxylic acid represented by the general formula (2) having a carboxy group used in the present invention, those known and used as polyvalent carboxylic acids can be preferably used. Specifically, adipic acid, Examples thereof include, but are not limited to, polycarboxylic acids such as sebacic acid and dodecanedioic acid.
In the esterification reaction, the carboxylic acid represented by the general formula (2) and the (meth) acrylic acid derivative represented by the general formula (1) may be reacted. The esterification reaction can be suitably carried out using an ester derivative of carboxylic acid. Examples of the ester group that can be used here include, for example, lower alkyl esters such as methyl ester and ethyl ester, but are not limited thereto, and publicly known and publicly available ester groups can be preferably used. .

Enzymes used in the present invention include the genus Aspergillus oryzae, the genus Achromobacter, the genus Bacillus, the genus Candida, the genus Chromobacter, the genus Fusarium, (Humicola), Hyphozyma genus, Pseudomonas genus, Rhizomucor genus, Rhizopus genus, or Thermomyces genus, preferably from Thermomyces genus microorganisms.
More specifically, preferred commercially available enzymes used in the present invention are listed as follows: lipase QL of Meisei Sangyo Co., Ltd., lipase PL of the same, lipase QLC, lipase QLG, lipase PLC, and lipase PLG of these immobilized enzymes. Other examples include Lipase PS manufactured by Amano Pharmaceutical Co., Ltd., Lipozyme RM-IM, Lipozyme TL-IM, Novozyme 435 manufactured by Novozymes. These enzymes may be used as a mixture, or may be changed according to the reaction process, or different enzymes may be added.

The amount of enzyme to be added to the reaction tank is preferably small from the viewpoint of labor and economical efficiency for removing the enzyme after the reaction, and from this viewpoint, a highly active enzyme is preferable. Specifically, 1000 U / g (activity measurement method by hydrolysis of fatty acid ester) or more is preferable, and 5000 U / g or more is preferable in the case of an enzyme that is difficult to disperse in a powder.
The form of the enzyme may be in the form of a solution, solid powder, or immobilized on a carrier, but is preferably solid powder or a carrier immobilized on a carrier that does not contain unnecessary components when the product is purified. In view of enzyme reuse, those immobilized on a carrier are more preferred.
The enzyme can be collected together with the unreacted raw material. The reaction solution after the reaction can be easily separated by decantation, centrifugation or filtration together with the enzyme and the unreacted raw material settled after standing or cooling. Cooling depends on the composition during the reaction, but is preferably room temperature or lower. The enzyme and unreacted raw material separated by filtration can be reused as they are, or once dried under heating and reduced pressure.

In the present invention, a highly active arming yeast in which a lipase having a high ester synthesis activity suitable for an ester reaction is expressed on the cell surface can also be preferably used.

The lipase arming yeast (B) used in the present invention has the following characteristics.
1. The lipase arming yeast (B) is a cell fusion of a haploid a cell arming yeast and a haploid α cell arming yeast,
2. The lipase arming yeast (B) is a protein that is displayed on the cell surface of one or both of the haploid a-cell arming yeast and the haploid α-cell arming yeast. Expressed on the cell surface by DNA having a sequence, a sequence encoding a part of the cell surface localized protein and a sequence encoding a GPI anchoring domain in this order;
3. The lipase arming yeast (B) is a protein that is displayed on the cell surface of one or both of the haploid a-cell arming yeast and the haploid α-cell arming yeast. A sequence, expressed on the cell surface by DNA having a sequence encoding a GPI anchoring domain in this order;
4). The lipase arming yeast (B) is a protein that is displayed on the cell surface of one or both of the haploid a-cell arming yeast and the haploid α-cell arming yeast. DNA having a sequence and a sequence encoding a sugar chain binding protein domain in this order, or a secretory signal sequence, a sequence encoding a sugar chain binding protein domain, and a structural gene sequence of the protein presented to the cell surface layer in this order It must be expressed on the cell surface by DNA.

The production of the arming yeast in which the lipase in the present invention is expressed on the cell surface can be carried out based on the published patent publications (JP-A-11-290078, WO2002 / 085935).

(Arming yeast)
In the present invention, the arming yeast is a cell surface localized protein that is fused with various functional proteins (enzymes, antigens, antibodies, reporter proteins, etc.) and peptides, and is displayed on the cell surface. A yeast cell that has a new function that is not present, or that has an enhanced function. The cell surface layer is a cell membrane, a cell wall, and a periplasm that is a space between them, and a yeast cell that uses these layers and satisfies the above requirements is called an arming yeast.

  The lipase in the present invention is as described below.

(Secretory enzyme)
A lipase is an enzyme having an activity capable of liberating fatty acids from fats and oils, and its origin is not particularly limited, but it is usually derived from fungi such as Rhizopus oryzae (hereinafter abbreviated as Rhizopus oryzae). Lipases derived from yeast such as lipase and Candida antarctica (hereinafter abbreviated as Candida antarctica) are preferably used.

(Cell surface localized protein)
In the present invention, the cell surface localized protein refers to a protein that is fixed on the cell surface of yeast and is present on the cell surface. For example, α- or a-agglutinin which is an aggregating protein, FLO protein, outer membrane protein OmpA of Escherichia coli and the like can be mentioned. In general, a cell surface localized protein has a secretory signal sequence on the N-terminal side and a GPI anchor attachment recognition signal sequence on the C-terminal side. Although it has the same secretory protein in that it has a secretory signal sequence, the cell surface localized protein is different from the secreted protein in that it is transported by being immobilized on the cell membrane via a GPI anchor. When the cell surface localized protein passes through the cell membrane, the GPI anchor adhesion recognition signal sequence is selectively cleaved, and is bound to the GPI anchor at the newly protruding C-terminal portion and fixed to the cell membrane. Thereafter, the base portion of the GPI anchor is cleaved by phosphatidylinositol-dependent phospholipase C (hereinafter abbreviated as PI-PLC). Subsequently, the protein cut off from the cell membrane is incorporated into the cell wall, fixed to the cell surface layer, and localized on the cell surface layer.

(Secretory signal sequence)
In the present invention, the secretory signal sequence is an amino acid sequence containing many amino acids rich in hydrophobicity, which is generally bound to the N-terminus of proteins secreted outside the cell. It is removed when it is secreted through the cell membrane to the outside of the cell (including the periplasm). Any secretory signal sequence can be used as long as it is a secretory signal sequence, and its origin is not limited. For example, a glucoamylase secretion signal sequence, a yeast α- or a-agglutinin secretion signal sequence, a lipase secretion signal sequence and the like are preferably used. If the lipase activity is not affected, a part or all of the secretory signal sequence may remain on the N-terminal side of the enzyme (Japanese Patent Laid-Open No. 11-290078, International Publication No. 2002/085935). (See brochure).

(GPI anchor and GPI anchoring domain)
The GPI anchor refers to a glycolipid called glycosylphosphatidylinositol (GPI) having ethanolamine phosphate-6 mannose α1-2 mannose α1-6 mannose α1-4 glucosamine α1-6 inositol phospholipid as a basic structure.

  The GPI anchoring domain is usually located at or near the C-terminus of the cell surface localized protein. For example, this corresponds to a sequence encoding a sequence of 320 amino acids from the C-terminus of α-agglutinin, and this sequence includes a GPI anchor recognition sequence that is recognized when a GPI anchor binds to a cell surface localized protein. In addition to the adhesion signal sequence, there are four sugar chain binding sites. After the base part of the GPI anchor is cleaved by PI-PLC, the sugar chain bound to these sugar chain binding sites and the polysaccharide constituting the cell wall are covalently bound, so that the C-terminal sequence part of α-agglutinin is Bound to the cell wall, α-agglutinin is retained on the cell surface.

(Glycan-binding protein domain)
In the present invention, the sugar chain-binding protein domain refers to a domain that has a plurality of sugar chains and can remain on the cell surface layer by interacting or entanglement with sugar chains in the cell wall. Examples include sugar chain binding sites such as lectins, and aggregation functional domains of aggregated proteins such as α-agglutinin, a-agglutinin, and FLO protein.
(Aggregating functional domain)
The aggregation functional domain of the cell surface localized protein refers to a domain that is located on the N-terminal side of the GPI anchoring domain, has a plurality of sugar chains, and is considered to be involved in aggregation.

In the present invention, the DNA for expressing lipase on the cell surface is preferably DNA having the following sequence.
(1) DNA having a secretory signal sequence, a lipase structural gene sequence, a sequence encoding a part of a cell surface localized protein, and a sequence encoding a GPI anchoring domain in this order
(2) DNA having a secretory signal sequence, a lipase structural gene sequence, and a sequence encoding a sugar chain binding protein domain in this order
(3) DNA having a secretory signal sequence, a sequence encoding a sugar chain binding protein domain, and a structural gene sequence of lipase in this order
(4) DNA having a secretory signal sequence, a lipase structural gene sequence, and a sequence encoding a GPI anchoring domain in this order
In the case of the above (2) and (3), it is preferable that the sugar chain binding protein domain is a part including at least the aggregation functional domain of the cell surface localized protein.

  In the case of the above (1) to (4), a structural gene sequence of lipase, a sequence encoding a part of a cell surface localized protein, a sequence encoding a sugar chain binding protein domain, or a GPI anchoring domain is encoded. A linker sequence having an appropriate length may be sandwiched between the sequences, and the type of amino acid is not limited, but a DNA sequence encoding 8 to 21 amino acids is preferred.

  The DNAs (1) to (4) can be synthesized using a conventionally known method. For example, in the case of the DNA of (1), the secretory signal sequence and the lipase structural gene sequence can be linked by using site-directed mutagenesis, and the accurate secretion signal sequence can be cleaved and highly active lipase can be obtained. Expression of B is possible. Furthermore, the sequence thus obtained may be combined with a sequence encoding a part of the cell surface localized protein and a GPI anchor attachment signal sequence. The binding can be performed using an appropriate restriction enzyme, linker or the like.

  The DNA may be in the form of a plasmid or in the form of being incorporated into a gene of a yeast of the genus Saccharomyces that serves as a host.

  Arming yeast can be obtained by introducing such DNA into Saccharomyces yeast.

The arming yeast obtained in this way is an arming yeast that has a higher activity for catalyzing the ester reaction than the conventional arming yeast and is more suitable for the ester reaction. Furthermore, since it has higher heat resistance than conventional arming yeasts and can catalyze ester reactions at high temperatures, it is possible to efficiently catalyze endothermic reactions such as ester synthesis reactions.
The use ratio of the (meth) acrylate represented by the general formula (1) and the aliphatic carboxylic acid is preferably equimolar or relative to the (meth) acrylate and the aliphatic carboxylic acid represented by the general formula (1). You may use it in the ratio of 1.1-1.5 times mole.
The amount of enzyme used in the reaction is 0.5 to 10% by mass, preferably 1 to 5% by mass with respect to the total mass of the (meth) acrylate represented by the general formula (3) and the fatty acid having a secondary hydroxyl group. %, More preferably 2 to 4% by mass.
In the reaction of the present invention, an enzyme reaction can be performed using the (meth) acrylate represented by the general formula (1) as a substrate and a reaction solvent, but an organic solvent that does not affect the enzyme activity is added. It can also be done. Such a reaction solvent is preferably not used, but may be added to the reaction system as needed for the purpose of improving the stirring efficiency of the reaction system and promoting dissolution of the substrate.

Examples of the solvent used include water, aqueous buffer solution, aqueous salt solution, methanol, ethanol, propanol, isopropanol, butanol, tertiary butanol, amyl alcohol, isoamyl alcohol, ethylene glycol, glycerin, monoglyme, diglyme, acetonitrile, methyl nitrate, Diethyl ether, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), diethyl ether, diisopropyl ether, THF, dioxane, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, benzene, toluene, xylene, dichloromethane , Chloroform, carbon tetrachloride, dichloroethane, trichloroethane, chlorobenzene, dichlorobenzene, trichlorobenzene, pyri Emissions, pentane, hexane, heptane, octane, isooctane, cyclohexane and the like are preferable, it may be a mixture of two or more thereof.
Water produced as a by-product in the reaction is preferably distilled off under reduced pressure by reducing the pressure in the reaction system and blowing in an inert gas such as nitrogen.

Considering the enzyme activity, it is preferable to control the water content of the reaction system, the water content is preferably 10 ppm to 10% (100,000 ppm), and for the enzyme activity, 10 ppm to 1% (10000 ppm) is preferably added. Is considered to be 10 ppm to 1000 ppm. These waters may be included in the enzyme to be added, or may be added separately to the reaction system, or small amounts of water may be added sequentially during the reaction.
The reaction temperature is preferably a high temperature in consideration of the solubility of the substrate and the reaction rate. On the other hand, the low temperature is preferable in consideration of the inactivation of the enzyme by heat, and it is necessary to satisfy the conflicting conditions. In the lipase used in the present invention, as a preferable reaction temperature, a temperature of 30 to 60 ° C. can be mentioned, and more preferably a temperature of 35 to 45 ° C. can be mentioned.
The reaction pressure is variable for the purpose of temperature control by boiling point, etc., and the reaction can be performed in the range of reduced pressure, pressurization, 0.13 to 1013 khPa (1 mmHg to 7600 mmHg), and usually 1.3 to 202.6 KPa (10 mmHg to 1520 mmHg). ) Is possible. The reaction time varies depending on the reaction system and is not generally defined. However, considering reaction efficiency and yield, it is generally preferably 3 hours to 48 hours, more preferably 5 hours to 30 hours. It is preferable to collect all unreacted raw materials and use them for re-reaction.

Although the reaction apparatus used for this invention is not specifically limited, In order to improve the dispersion | distribution collision property of a substrate and an enzyme, reaction with the reaction tank which has a stirring apparatus is preferable. That is, water distilled from the reaction tank is passed through a condenser, and then the agglomerated water is passed through a byproduct removal tank filled with a filler such as molecular sieves, and water is adsorbed and removed by molecular sieves.
As the filler to be filled in the by-product removal tank, it is preferable to select a filler that does not adsorb (meth) acrylate and the solvent. Molecular sieves, silica gel, calcium chloride, potassium chloride, sodium chloride, magnesium oxide, magnesium sulfide, Examples include potassium carbonate and sodium sulfate. The molecular sieve needs to be appropriately changed depending on the type of by-product, but the 3A type is preferable if the by-product is water or methanol, and the 4A type is preferable if these are ethanol or ethanol. A by-product removal tank having a volume of 1% to 100% of the reaction tank is sufficient.

The molecular sieve can adsorb water having a weight of about 30% of its weight, but is preferably filled with an excess amount from the viewpoint of reaction efficiency. It is necessary to regenerate the filler during or after the reaction, and examples of the regeneration method include washing, heating, and decompression. In general, after drying with water and alcohol, drying by heating and drying under reduced pressure are performed. The temperature is preferably 100 ° C. or higher, and rapid regeneration can be expected at 180 ° C. or higher.
In order to reduce the scale of the by-product removal tank, it is efficient to regenerate the filler during the reaction. For example, two or more removal tanks are used in parallel, and the operation tanks are switched to operate continuously. It is preferable. It is also preferable that the by-product removal tank has a structure like a Soxhlet extractor. Moreover, it is also possible not to have a by-product removal tank, but to put these packing materials into the reaction tank directly to react and to remove them from the reaction product after the reaction.
As the stirring device, screw blades, helical blades, fiddler blades, turbine blades, paddle blades, and the like can be used, and it is preferable that homogeneous stirring is possible. The stirring rotation speed depends on the stirring efficiency and stirring power, and therefore depends on the stirring blade, the scale of the reaction tank, etc., but when the substrate is heterogeneous, a low speed is preferable as long as precipitation does not occur.

Hereinafter, the present invention will be described specifically by way of examples.

(Example 1) Reaction of caprylic acid with HEMA Three-way cock (one is connected to a vacuum pump through a cooling trap, the other is connected to a vacuum gauge) and a gas introduction pipe (coupled to a gas flow rate controller) Is attached to the three-necked flask
Caprylic acid 28.8g, 0.20 ⁰ mol
2-hydroxyethyl methacrylate 28.6 g, 0.22 ⁰ mol
Methoxyhydroquinone 9mg
1.7g of water
Lipozyme RM-IM 1.7 g (3% by weight based on the total amount of monomers)
And heated to 45 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. After 4 hours, the pressure inside the reaction system was reduced (3.5 kPa) and dry air was supplied. The reaction conversion rate after 24 hours from the start of the reaction was> 97% (GC peak). The pressure reduction and nitrogen blowing in the reaction system were stopped, and the immobilized enzyme was separated by filtration (using Advantech production filter paper No. 28-3). A transparent liquid (purity 93% (GC peak ratio), Hazen color number 20) was obtained at room temperature.

In the present invention, reaction tracking and purity determination were performed by gas chromatogram.
<Gas chromatogram conditions>
Measuring equipment: Shimadzu GC-2010
Column: Capillary column TC-5 (0.32 mm ID × 30 m, df = 0.25 μm, manufactured by GL Sciences)
Injection method: Split method (30: 1)
Injection temperature: 300 ° C
Detection method: FID
Detector temperature: 320 ° C
Column temperature: 100 ° C. (hold for 2 minutes) → 20 ° C./minute→300° C. (hold for 13 minutes)
Moreover, the compound identification of the mixture was confirmed by mass spectrometry (GC-MS).
<GC-MS conditions>
Measuring instrument: JEOL JMS-K9 (quadrupole type)
Column: Capillary column DB-5 ms (0.25 mm ID × 30 m, df = 0.25 μm, manufactured by Agilent Technologies)
Injection method: Split method (30: 1)
Injection temperature: 300 ° C
Column temperature: 50 ° C. (2 minutes hold) → 15 ° C./min→300° C.
Ionization method: EI or CI (isobutane)

(Example 2) Reaction of methyl caprylate and HEMA Three-way cock (one is connected to a vacuum pump through a cooling trap, the other is connected to a vacuum gauge) and a gas introduction pipe (coupled to a gas flow control controller) To the three-necked flask with
Methyl caprylate 34.8gl, 0.22 ⁰ mol
2-hydroxyethyl methacrylate 31.5 g, 0.24 ² mol
Methoxyhydroquinone 7mg
Lipozyme RM-IM 3.3 g (5% by weight based on the total amount of monomers)
And heated to 45 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. After 30 minutes, the pressure in the reaction system was reduced (2.5 kPa), and dry air was supplied. 8 hours after the start of the reaction, the degree of vacuum in the reaction system was increased to 1.5 kPa, and then 24 hours after the start of the reaction, the reaction was continued to increase to 0.6 kPa. The reaction conversion rate after 24 hours from the start of the reaction was 93%, and the conversion rate after 36 hours was 97%. 72 hours after the start of the reaction, after confirming the disappearance of the methyl caprylate peak, the pressure reduction in the reaction system and the blowing of air were stopped, and immobilization was performed by filtration (using filter paper No. 28-3 for production manufactured by Advantech). The enzyme was filtered off. A clear liquid was obtained at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

Comparative Example 1 Reaction of Caprylic Acid with HEMA (Chemical Synthesis Example)
In a three-necked flask equipped with a decanter and a gas introduction tube,
Caprylic acid 28.8g, 0.20 ⁰ mol
2-hydroxyethyl methacrylate 31.2 g, 0.24 ⁰ mol
Methoxyhydroquinone 0.1g
p-Toluenesulfonic acid 1.5g
Toluene 240.0g
And heated to 125-130 ° C. in an oil bath. For stirring, a magnetic stirrer was used, and dry air was supplied by bubbling. Formation of condensed water was confirmed with the reflux of toluene. Six hours after the start of the reaction, the generation of condensed water was stopped, and the reaction was stopped because no progress of the reaction was observed from the reaction tracking results. The conversion rate at this time was 91%. The reaction mixture was washed with 5% aqueous sodium carbonate solution and then with saturated brine and distilled water. The reaction mixture was dried over anhydrous magnesium sulfate, and then toluene was distilled off to obtain a pale yellow liquid (purity 85% (GC peak ratio), Hazen color number 150).
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

(Comparative Example 2) Reaction of caprylic acid with HEMA (Experimental example using lipase not showing selectivity)
The same reaction as in Example 1 was performed except that Novozyme 435 (an immobilized enzyme of lipase B derived from Candida antarctica) was used instead of lipozyme RM-IM as an enzyme catalyst.

As a result of reaction tracking by GC analysis, a plurality of peaks other than the peak of the target product (Rt: 8.9 minutes) were generated as the reaction progressed. The reaction conversion after 24 hours from the start of the reaction was> 98%. The pressure reduction and nitrogen blowing in the reaction system were stopped, and the immobilized enzyme was separated by filtration (using Advantech production filter paper No. 28-3). A clear liquid was obtained at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

  When GC analysis was performed on the obtained compound, the purity of the target compound was 51% (GC peak ratio).

(Analysis of Example 1 and Comparative Examples 1 and 2)
The reaction product was analyzed by GC analysis and GC mass spectrometry, and the reaction selectivity (purity) was calculated from the product identification and the GC peak area.

(In the table, EGDMA represents ethylene glycol dimethacrylate, HO-EG-C8 represents ethylene glycol monocaprylate, and C8-EG-C8 represents ethylene glycol dicaprylate.)

From the results of Example 1 and Comparative Examples 1 and 2, in Example 1, a high-purity and uncolored target product was obtained.

(Example 3) Reaction of caproic acid with HEMA Three-way cock (one is connected to a vacuum pump through a cooling trap, the other is connected to a vacuum gauge) and a gas introduction pipe (coupled to a gas flow rate controller) Is attached to the three-necked flask
Caproic acid 28.3, 0.25 ⁰ mol
2-Hydroxyethyl methacrylate 35.8 g, 0.27 ⁵ mol
Methoxyhydroquinone 6mg
3.2g of water
Lipozyme RM-IM 1.9 g (3% by weight based on the total amount of monomers)
And heated to 45 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. After 1.5 hours, the pressure in the reaction system was reduced (3.5 kPa), and dry air was supplied. After 8.5 hours from the start of the reaction, the degree of vacuum in the reaction system was increased to 5.0 kPa, and the reaction was continued. 48 hours after the start of the reaction, the pressure reduction and air blowing in the reaction system were stopped, and the immobilized enzyme was separated by filtration (using production paper No. 28-3 manufactured by Advantech). A clear liquid was obtained at room temperature. The purity of the target product was 90% (GC peak ratio).
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

(Example 4) Reaction of palmitic acid with HEMA Three-way cock (one is connected to a vacuum pump through a cooling trap, the other is connected to a vacuum gauge) and a gas introduction pipe (coupled to a gas flow rate controller) Is attached to the three-necked flask
Palmitic acid 38.5g, 0.15 ⁰ mol
2-Hydroxyethyl methacrylate 21.5 g, 0.16 ⁵ mol
Methoxyhydroquinone 7mg
Lipozyme RM-IM 1.8 g (3% by weight based on the total amount of monomers)
And heated to 60 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. At the start of the reaction, palmitic acid was in a partially dissolved suspension state. After 15 minutes, the pressure in the reaction system was reduced (3.5 kPa), and dry air was supplied. All the solid palmitic acid dissolved as the reaction proceeded. 20 hours after the start of the reaction (reaction conversion> 98%), the pressure reduction and air blowing in the reaction system were stopped, and the immobilized enzyme was separated by filtration (using Advantech production filter paper No. 28-3). did. The purity of the product was 85% (GC peak ratio).
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

(Example 5) Reaction of oleic acid with HEMA Three-way cock (one is connected to a vacuum pump through a cooling trap, the other is connected to a vacuum gauge) and a gas introduction pipe (coupled to a gas flow rate controller) Is attached to the three-necked flask
42.4g Oleic acid, 0.15 ⁰ mol
2-Hydroxyethyl methacrylate 21.5 g, 0.16 ⁵ mol
Methoxyhydroquinone 12.7mg
Lipozyme RM-IM 2.0 g (3% by weight based on the total amount of monomers)
And heated to 45 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. After 30 minutes, the reaction system was depressurized (3.5 kPa) and condensed water was removed while supplying dry air. Two hours after the start of the reaction, the degree of vacuum in the reaction system was increased to 2.0 kPa, and the reaction was continued. The reaction conversion after 18 hours from the start of the reaction was> 96%. The pressure reduction and air blowing in the reaction system were stopped, and the immobilized enzyme was separated by filtration (using Advantech filter paper No. 28-3 for production). A pale yellow liquid (Hazen color number <1) was obtained at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

(Comparative Example 3) Reaction of oleic acid with HEMA (chemical synthesis method)
To a four-necked flask equipped with a decanter and a gas introduction tube.
42.4g Oleic acid, 0.15 ⁰ mol
2-Hydroxyethyl methacrylate 23.4 g, 0.18 ⁰ mol
Methoxyhydroquinone 100mg
Toluene 270.0g
2.0 g of p-toluenesulfonic acid monohydrate (3% by weight based on the total amount of monomers)
And heated to 125-130 ° C. in an oil bath. For stirring, a magnetic stirrer was used, and dry air was supplied by bubbling. Formation of condensed water was confirmed with the reflux of toluene, but the reaction solution turned brown. Four hours after the start of the reaction, the generation of condensed water was stopped, and the reaction was stopped because no progress of the reaction was observed from the reaction tracking results. The conversion rate at this time was 76%. Attempts were made to wash the reaction mixture with alkali, but emulsification was severe and washing with water was difficult. Further, decolorization from the reaction mixture by alkali and water washing was not possible, and the obtained compound was a brown liquid (Hazen color number> 18).
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

Example 6 Reaction of 10-Undecenoic Acid with HEMA Three-way cock (one coupled to a vacuum pump through a cooling trap and the other coupled to a vacuum gauge) and a gas inlet tube (coupled to a gas flow control controller) To the three-necked flask equipped with
10-Undecenoic acid 36.9 g, 0.20 ⁰ mol
2-hydroxyethyl methacrylate 28.6 g, 0.22 ⁰ mol
Methoxyhydroquinone 7mg
1.3g of water
Lipozyme RM-IM 2.0 g (3% by weight based on the total amount of monomers)
And heated to 45 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. After 4 hours, the pressure in the reaction system was reduced (6.0 kPa), and dry air was supplied. Seven hours after the start of the reaction, the degree of vacuum in the reaction system was increased to 3.5 kPa, and the reaction was continued. 28 hours after the start of the reaction (reaction conversion rate 97%), the pressure reduction and air blowing in the reaction system were stopped, and the immobilized enzyme was separated by filtration (using Advantech production filter paper No. 28-3). . A clear liquid (purity 90%) was obtained at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

Example 7 Reaction of Dehydrated Castor Oil Fatty Acid DCO / FA with HEMA (Synthesis Example of Oxygen-Stable Compound)
A three-necked flask fitted with a three-way cock (one connected to a vacuum pump through a cooling trap and the other to a vacuum gauge) and a gas inlet tube (connected to a gas flow control controller)
DCO / FA42.4g, 0.15 ⁰ mol
(Calculated from average value)
2-Hydroxyethyl methacrylate 21.5 g, 0.16 ⁵ mol
Methoxyhydroquinone 7mg
Lipozyme RM-IM 1.9 g (3% by weight based on the total amount of monomers)
And heated to 45 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. After 15 minutes, the pressure in the reaction system was reduced (2.5 kPa), and dry nitrogen was supplied. 2.5 hours after the start of the reaction, the degree of vacuum in the reaction system was increased to 1.5 kPa, and the reaction was continued. Twenty hours after the start of the reaction, the pressure reduction and nitrogen blowing in the reaction system were stopped, and the immobilized enzyme was separated by filtration (using production paper No. 28-3 manufactured by Advantech). A pale yellow transparent liquid was obtained at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

  GC / MS analysis of the obtained reaction product showed that it was a mixture produced by selective esterification reaction between each peak of the raw material DCO / FA (mixture) and HEMA (FIG. 1 (raw material) And FIG. 2 (product) shows a GC chart.)

Comparative Example 4 Reaction of Dehydrated Castor Oil Fatty Acid DCO / FA and HEMA (Chemical Synthesis Example of Oxygen-Stable Compound)
In the flask
DCO / FA42.4g, 0.15 ⁰ mol
2-Hydroxyethyl methacrylate 21.5 g, 0.16 ⁵ mol
Methoxyhydroquinone 100mg
Toluene 270.0g
It was placed. Warm to 100 ° C. in an oil bath. For stirring, a magnetic stirrer was used, and dry air was supplied by bubbling. After 2.5 hours from the start of the reaction, the reaction was stopped because a precipitate was formed.

From the results of Example 7 and Comparative Example 4, the present invention can also be applied to reactions using dehydrated castor oil fatty acid and the like unstable to oxygen and heat as raw materials.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

(Example 8) Reaction of capric acid with HEA Three-way cock (one is connected to a vacuum pump through a cooling trap, the other is connected to a vacuum gauge) and a gas introduction pipe (coupled to a gas flow rate controller) Is attached to the three-necked flask
Capric acid 37.9g, 0.22 ⁰ mol
2-Hydroxyethyl acrylate 26.8 g, 0.23 ¹ mol
Methoxyhydroquinone 8mg
1.3g of water
Lipozyme RM-IM 3.2 g (5% by weight based on the total amount of monomers)
And heated to 40 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. After 2.5 hours, the pressure in the reaction system was reduced (5.0 kPa), and dry air was supplied. Six hours after the start of the reaction, the degree of vacuum in the reaction system was temporarily stopped. After 10 hours from the start of the reaction (conversion rate 90%), the pressure reduction in the reaction system and the blowing of air were stopped, and the immobilized enzyme was separated by filtration (using Advantech production filter paper No. 28-3). A transparent liquid (purity 93% (GC peak ratio)) was obtained at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

(Example 9) Reaction of palmitic acid and HEA Three-way cock (one is connected to a vacuum pump through a cooling trap, the other is connected to a vacuum gauge) and a gas introduction pipe (coupled to a gas flow rate controller) Is attached to the three-necked flask
Palmitic acid 153.9g, 0.60 ⁰ mol
2-hydroxyethyl acrylate 76.6 g, 0.63 ⁰ mol
Methoxyhydroquinone 31mg
Lipozyme RM-IM 6.9 g (3% by weight based on the total amount of monomers)
And heated to 60 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. At the start of the reaction, palmitic acid was in a partially dissolved suspension state. After 1 hour, the pressure in the reaction system was reduced (5.0 kPa), and dry air was supplied. All the solid palmitic acid dissolved as the reaction proceeded. Two hours after the start of the reaction, the degree of vacuum in the reaction system was increased to 3.5 kPa, and the reaction was continued. 24 hours after the start of the reaction (reaction conversion> 98%), the pressure reduction and air blowing in the reaction system were stopped, and the immobilized enzyme was filtered off by filtration (using production paper No. 28-3 manufactured by Advantech). did.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

  The following purification was performed to remove a small amount of high melting point by-product.

  The reaction mixture was dispersed in methanol, and insoluble materials were removed by filtration. The oily liquid obtained after concentrating the filtrate was poured into ice water and dispersed. The oily liquid solidified. The white solid was separated by filtration and dried to obtain white crystals (purity 95% (GC peak ratio), melting point 27.9 ° C.) at room temperature.

(Example 10) Reaction of stearic acid with HEA (using solvent)
Dean-Stark trap with a three-way cock (one connected to a vacuum pump via a cooling trap and the other to a vacuum gauge) and a three-neck fitted with a gas inlet tube (coupled to a gas flow control controller) Into the flask,
Stearic acid 128.0 g, 0.45 ⁰ mol
2-Hydroxyethyl acrylate 57.5 g, 0.49 ⁵ mol
Methoxyhydroquinone 38mg
Lipozyme RM-IM 3.7 g (2% by weight based on the total amount of monomers)
Toluene 100g
And heated to 45 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. After 27.5 hours, the pressure in the reaction system was reduced (50 kPa) and dry air was supplied. After 32 hours from the start of the reaction, the reaction temperature was raised to 65 ° C., and the reaction was continued while removing toluene. 48 hours after the start of the reaction, the pressure reduction and air blowing in the reaction system were stopped, and the immobilized enzyme was separated by filtration (using production paper No. 28-3 manufactured by Advantech).
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

In order to remove unreacted raw materials, the following purification was performed.
The reaction mixture was dissolved in toluene and washed twice with 5% aqueous sodium hydrogen carbonate solution, saturated brine and distilled water. After drying over anhydrous magnesium sulfate, toluene was distilled off under reduced pressure to obtain white crystals (purity 96% (GC peak ratio), melting point: 34.5 ° C.) at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

(Example 11) Reaction of capric acid with HBMA Three-way cock (one is connected to a vacuum pump through a cooling trap and the other is connected to a vacuum gauge) and a gas introduction pipe (coupled to a gas flow rate controller) Is attached to the three-necked flask
Capric acid 25.8g, 0.15 ⁰ mol
2-Hydroxybutyl methacrylate 24.9 g, 0.15 ⁸ mol
Methoxyhydroquinone 5mg
1g of water
Lipozyme RM-IM 1.5 g (3% by weight based on the total amount of monomers)
And heated to 40 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. After 5 hours, the pressure in the reaction system was reduced (8.0 kPa) and dry air was supplied. 30 hours after the start of the reaction, the decompression was stopped and the reaction was continued as it was. 48 hours after the start of the reaction, the inside of the reaction system was again decompressed (5.0 kPa) and supplied with dry air. 58 hours after the start of the reaction, the degree of vacuum in the reaction system was increased to 2.5 kPa, and the reaction was continued. 74 hours after the start of the reaction, the pressure reduction and air blowing in the reaction system were stopped, and the immobilized enzyme was separated by filtration (using filter paper No. 28-3 for production manufactured by Advantech). A clear liquid was obtained at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

(Example 12) Reaction of capric acid with HEAAm Three-way cock (one is connected to a vacuum pump through a cooling trap and the other is connected to a vacuum gauge) and a gas introduction pipe (coupled to a gas flow rate controller) Is attached to the three-necked flask
Capric acid 43.1 g, 0.25 ⁰ mol
2-hydroxyethyl methacrylate 30.2 g, 0.25 ⁰ mol
Methoxyhydroquinone 43mg
Novozyme 435 1.5 g (2% by weight based on the total amount of monomers)
And heated to 45 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. After 15 minutes, the pressure in the reaction system was reduced (3.5 kPa), and dry air was supplied. Four hours after the start of the reaction, the degree of vacuum in the reaction system was increased to 2.5 kPa, and the reaction was continued. The temperature was raised to 60 ° C. 23 hours after the start of the reaction for crystallization. Twenty-four hours after the start of the reaction, the pressure reduction in the reaction system and the blowing of air were stopped, and the immobilized enzyme was separated by filtration (using filter paper No. 28-3 for production manufactured by Advantech). A white solid (purity 90% (GC peak ratio), melting point: 44.7 ° C.) was obtained at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

(Example 13) Reaction of palmitic acid with HEAAm Three-way cock (one is connected to a vacuum pump through a cooling trap and the other is connected to a vacuum gauge) and a gas introduction pipe (coupled to a gas flow rate controller) Is attached to the three-necked flask
Palmitic acid 153.9g, 0.60 ⁰ mol
2-hydroxyethyl acrylamide 72.5 g, 0.63 ⁰ mol
Methoxyhydroquinone 154mg
Novozyme 435 4.5g (2% by weight based on the total amount of monomers)
And heated to 65 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. After 15 minutes, the pressure in the reaction system was reduced (15.0 kPa) and dry air was supplied. Four hours after the start of the reaction, the reaction temperature was raised to 70 ° C. for crystal precipitation. After 8 hours from the start of the reaction, the entire reaction system was crystallized, so 100 ml of toluene was added, and the immobilized enzyme was separated by filtration (using Advantech filter paper No. 28-3 for production). To the obtained filtrate was further added 500 ml of toluene, and the mixture was allowed to cool to room temperature to obtain precipitated white crystals (purity 94% (GC peak ratio), melting point 73.9 ° C.).
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

Example 14 Reaction of 10-Undecenoic Acid with HEAAm Three-way cock (one coupled to a vacuum pump through a cooling trap and the other to a vacuum gauge) and a gas inlet tube (coupled to a gas flow control controller) To the three-necked flask equipped with
10-undecenoic acid 46.1 g, 0.25 ⁰ mol
2-hydroxyethylacrylamide 30.2 g, 0.26 ³ mol
Methoxyhydroquinone 46mg
Novozyme 435 0.8g (1% by weight based on the total amount of monomers)
And heated to 45 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. After 15 minutes, the pressure in the reaction system was reduced (5 kPa), and dry air was supplied. Two hours after the start of the reaction, the degree of vacuum in the reaction system was increased to 2.5 kPa, and the reaction was continued. Twenty-four hours after the start of the reaction, the pressure reduction in the reaction system and the blowing of air were stopped, and the immobilized enzyme was separated by filtration (using filter paper No. 28-3 for production manufactured by Advantech). Solidified at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

(Example 15) Reaction of divinyl adipate and HEMA (reaction using lipozyme TL-IM)
A three-necked flask fitted with a three-way cock (one connected to a vacuum pump through a cooling trap and the other to a vacuum gauge) and a gas inlet tube (connected to a gas flow control controller)
Divinyl adipate 99.1 g, 0.50 ¹ mol
2-hydroxyethyl methacrylate 136.6 g, 1.05 ⁰ mol
Methoxyhydroquinone 50mg
Lipozyme TL-IM 7.1 g (3% by weight based on the total amount of monomers)
And heated to 40 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. After 15 minutes, the inside of the reaction system was depressurized (30 kPa), and dry air was supplied. Four hours after the start of the reaction, the degree of vacuum in the reaction system was increased to 25 kPa, and then to 8.5 kPa after 10 hours, and the reaction was continued. 28 hours after the start of the reaction, the pressure reduction and air blowing in the reaction system were stopped, and the immobilized enzyme was separated by filtration (using production paper No. 28-3 manufactured by Advantech). The obtained filtrate was washed with saturated brine, and then a light yellow transparent liquid (purity 92% (GC peak ratio)) was obtained at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

(Example 16) Reaction of divinyl adipate with HEMA (reaction using Novozyme 435)
A three-necked flask fitted with a three-way cock (one connected to a vacuum pump through a cooling trap and the other to a vacuum gauge) and a gas inlet tube (connected to a gas flow control controller)
Divinyl adipate 19.8 g, 0.10 ⁰ mol
2-Hydroxyethyl methacrylate 27.3 g, 0.21 ⁰ mol
Methoxyhydroquinone 23mg
Novozyme 435 1.4 g (3% by weight based on the total amount of monomers)
And heated to 40 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. The inside of the reaction system was depressurized (10 kPa), and dry air was supplied. Two hours after the start of the reaction, the pressure reduction and air blowing in the reaction system were stopped, and the immobilized enzyme was separated by filtration (using Advantech production filter paper No. 28-3). A pale yellow transparent liquid (purity 86% (GC peak ratio)) was obtained at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

(Example 17) Reaction of sebacic acid with HEMA Three-way cock (one is connected to a vacuum pump through a cooling trap and the other is connected to a vacuum gauge) and a gas introduction pipe (coupled to a gas flow rate controller) Is attached to the three-necked flask
Sebacic acid 40.5 g, 0.20 ⁰ mol
2-Hydroxyethyl methacrylate 53.4 g, 0.41 ⁰ mol
Methoxyhydroquinone 8mg
1.9g of water
Lipozyme RM-IM 4.7 g (5% by weight based on the total amount of monomers)
And heated to 55 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. At the start of the reaction, sebacic acid was in a partially dissolved suspension state. After 3 hours, the inside of the reaction system was depressurized (5.0 kPa), and dry air was supplied. As the reaction proceeded, all solid sebacic acid dissolved. 24 hours after the start of the reaction, the degree of vacuum in the reaction system was increased to 2.5 kPa, and the reaction was continued. After 30 hours from the start of the reaction, the pressure reduction and air blowing in the reaction system were stopped, and the immobilized enzyme was separated by filtration (using Advantech production filter paper No. 28-3). A transparent liquid (purity 90% (GC peak ratio)) was obtained at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

(Example 18) Reaction of dodecanedioic acid with HEMA Three-way cock (one is connected to a vacuum pump through a cooling trap and the other is connected to a vacuum gauge) and a gas introduction pipe (coupled to a gas flow control controller) To the three-necked flask with
Dodecanedioic acid 34.6g, 0.15 ⁰ mol
2-hydroxyethyl methacrylate 40.0 g, 0.30 ⁸ mol
Methoxyhydroquinone 7mg
1.5g of water
Lipozyme RM-IM 3.7 g (5% by weight based on the total amount of monomers)
And heated to 50 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. At the start of the reaction, dodecanedioic acid was in a partially dissolved suspension state. After 3 hours, the inside of the reaction system was depressurized (5.0 kPa), and dry air was supplied. As the reaction proceeded, all solid dodecanedioic acid dissolved. Twenty-five hours after the start of the reaction, the pressure reduction and air blowing in the reaction system were stopped, and the immobilized enzyme was removed by filtration (using Advantech production filter paper No. 28-3). A transparent liquid (purity 85% (GC peak ratio)) was obtained at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

(Example 19) Reaction of methyl caprylate and HEMA Three-way cock (one is connected to a vacuum pump through a cooling trap, the other is connected to a vacuum gauge) and a gas introduction pipe (coupled to a gas flow control controller) To the three-necked flask with
Caprylic acid 43.2g, 0.30 ⁰ mol
2-hydroxyethyl methacrylate 42.9 g, 0.33 ⁰ mol
Methoxyhydroquinone 15mg
CALB-arming yeast 7.5 g (8.7% by weight based on the total amount of monomers)
And heated to 40 ° C. in an oil bath. The reaction was stirred using a mechanical stirrer equipped with a stirring blade. After 3 hours, the pressure in the reaction system was reduced (5 kPa) and dry air was supplied. 24 hours after the start of the reaction, the degree of vacuum in the reaction system was increased to 1 kPa, and the reaction was continued. The reaction conversion after 46 hours from the start of the reaction was 96.6%. At this point, pressure reduction and air blowing in the reaction system were stopped, and the CALB-arming yeast was separated by filtration (using Advantech filter paper No. 28-3 for production). A clear liquid was obtained at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

(Example 20) Reaction of 12-hydroxystearic acid and HEMA Three-way cock (one is connected to a vacuum pump through a cooling trap, the other is connected to a vacuum gauge) and a gas introduction pipe (to a gas flow rate controller) In a three-necked flask equipped with
12-hydroxystearic acid 150.2 g, 0.500 mol
Methoxyhydroquinone 45mg
The 12-hydroxystearic acid was melted in an oil bath (90 ° C.) and then allowed to cool to 80 ° C.
2-hydroxyethyl methacrylate 71.6 g, 0.550 mol
6.7 g of lipozyme RM-IM (3% by weight based on the total amount of monomers)
To start the reaction. The reaction temperature was gradually cooled to 70 ° C., and after 2 hours, the reaction system was depressurized (5.0 kPa) and dry air was supplied. After 20 hours from the start of the reaction, the pressure was reduced to 3.5 kPa and after 30 hours, the reaction was continued to 2.0 kPa (reaction conversion 96%). The pressure reduction and air blowing in the reaction system were stopped, and the immobilized enzyme was separated by filtration (using Advantech filter paper No. 28-3 for production). A white waxy solid (yield 187.3 g, yield 88%, purity 92% (GC peak ratio)) was obtained at room temperature.
In the same manner as in Example 1, reaction tracking and purity determination were performed by gas chromatogram, and compound identification of the mixture was confirmed by mass spectrometry (GC-MS).

The preparation method of the arming yeast used in the above Example is described below.
(Production Example 1) <Preparation of CALB surface expression plasmid>
(1-A. Acquisition of Candida antarctica-derived lipase B gene)
Candida antarctica-derived lipase B (CALB) gene was obtained as follows.

That is, PCR amplification was carried out using BglII and XhoI with primers composed of the nucleotide sequences shown in SEQ ID NOs: 1 and 2, synthesized with a DNA synthesizer by a conventionally known method using the Candida antarctica CBS6678 genome as a template. Cleavage gave a BglII-XhoI fragment (BglII-XhoI ProCALB CBS6678 fragment) approximately 1000 bp long. This CALB is described in the literature of Uppberg et al. (Structure, 2: 293 (1994)), and compared with the most widely known amino acid sequence of lipase B derived from Candida antarctica LF058 (CALB WT). Is mutated. That is, 25th alanine is threonine, 28th serine is threonine, 31st serine is threonine, 46th glutamine is glycine, 89th alanine is threonine, 97th asparagine is arginine, 286 The second valine was a characteristic CALB, each mutated to isoleucine.
(1-B. Acquisition of CALB gene transfer plasmid)
In order to efficiently introduce mutations into the CALB gene, the CALB CBS6678 gene obtained in 1-A above was once introduced into a small-size plasmid.

  That is, plasmid pUC19 (manufactured by TaKaRa BIO) was digested with BamHI and SalI, and the BglII-XhoI ProCALB CBS6678 fragment obtained in A above was inserted to obtain plasmid pUC19-ProCALB CBS6678. A schematic diagram of the fabrication is shown in FIG.

(1-C. Acquisition of CALB1 gene and CALB1 introduction plasmid)
PCR amplification is performed using the plasmid pUC19-ProCALB CBS6678 obtained in 1-B above as a template and a primer consisting of the nucleotide sequences shown in SEQ ID NOs: 3 and 4 synthesized by a DNA synthesizer by a conventionally known method. To introduce a point mutation into CALB CBS6678, which includes the CALB1 gene in which the 25th threonine from the amino terminus in CALB CBS6678 is substituted with alanine, the 28th threonine with serine, and the 31st threonine with serine. The plasmid pUC19-ProCALB1 was obtained.

(1-D. Acquisition of CALB2 gene and CALB2 introduction plasmid)
By performing PCR amplification using the plasmid pUC19-ProCALB1 obtained in 1-C above as a template and synthesizing with a DNA synthesizer by a conventionally known method using primers consisting of the nucleotide sequences shown in SEQ ID NOs: 5 and 6 Then, a point mutation was introduced into CALB1, and a plasmid pUC19-ProCALB2 containing CALB2 gene in which the 46th glycine from the amino terminus in CALB1 was replaced with glutamine was obtained.

(1-E. Acquisition of linker-introduced CALB gene)
Using each CALB2 introduced plasmid obtained in 1-D above as a template, PCR amplification was performed using primers composed of the nucleotide sequences shown in SEQ ID NOs: 7 and 8, synthesized by a DNA synthesizer by a conventionally known method, Cleavage with BglII and XhoI gave a BglII-XhoI FLAG-ProCALB2 fragment of approximately 1000 bp in length. The BglII-XhoI FLAG-ProCALB2 fragment has the FLAG tag, the lipase prosequence, and the mature protein sequence of CALB2. Here, the pro sequence is a sequence required to form the lipase three-dimensional structure, the FLAG tag is a linker sequence, and the mature protein sequence is actually a lipase catalyst. A sequence having a reaction function.

(1-F. Preparation of CALB surface expression cassette)
The CALB surface expression cassette has an expression promoter sequence and a terminator sequence, and a short-type FLO1 anchor gene, which is a FLO1 derivative, and a linker-introduced CALB gene obtained in the above 1-E connected between them. DNA sequence. FIG. 2 shows a schematic diagram of production in which the following operations were performed in order to produce the CALB surface expression cassette.

  Specifically, the plasmid pWIFS (T. Matsumoto et al., Appl. Environ. Microbiol., 68: 4517 (2002)) was cleaved with BglII and XhoI, and the BglII-XhoI FLAG-ProCALB2 fragment obtained in 1-E above was inserted. Thus, pWIFS-FLAG-ProCALB2 was obtained. This was cleaved with BssHII to obtain a CALB surface expression cassette of about 6300 bp.

(Preparation of 1-G.CALB surface expression plasmid)
A plasmid having the target DNA can be obtained by introducing the CALB surface expression cassette obtained in 1-F above into a plasmid having various selection markers. A schematic diagram of the fabrication is shown in FIG.

  Specifically, plasmids pRS402 (ATCC87477), pRS403 (ATCC87514), pRS404 (ATCC87515), pRS405 (ATCC87516), and pRS402 (ATCC87517) were each cut with BssHII, and the CALB surface expression cassette obtained in F above was inserted into pRSAIFS- FLAG-ProCALB2, pRSHIFS-FLAG-ProCALB2, pRSWIFS-FLAG-ProCALB2, pRSLIFS-FLAG-ProCALB2, and pRSUIFS-FLAG-ProCALB2 were obtained.

(Production Example 2) <Preparation of genome-integrated diploid CALB-arming yeast>
(2-A. Introduction of genes into Saccharomyces yeasts)
The CALB surface expression plasmid obtained in 1-G above was cleaved at the restriction enzyme sites present in the selectable markers using the restriction enzymes shown in Table 1, respectively. Then, using YEAST MAKER ^™ (Clontech Laboratories Inc. USA), Saccharomyces cerevisiae (hereinafter abbreviated as Saccharomyces cerevisiae) YPH499 (MATa, ura2, le2) , PRSUIFS-FLAG-ProCALB2, pRSLIFS-FLAG-ProCALB2, pRSHIFS-FLAG-ProCALB2, pRSAIFS-FLAG-ProCALB2 in this order, Saccharomyces cerevisiae YPH500 (MATalpha, ura3, lys2, ras2, asp2, ras2, asp2, , PRSUIFS-FLAG-ProCA B2, the pRSLIFS-FLAG-ProCALB2, pRSWIFS-FLAG-ProCALB2, pRSAIFS-FLAG-ProCALB2 in this order, were introduced into the respective genome. In each transformation selection, amino acids as well as nucleic acids (0.002% L-histidine, 0.01% L-leucine, 0.002% L-tryptophan, 0.002% L-lysine, 0.002% adenine, The culture was performed using SD agar medium (2% glucose, 0.67% Yeast Nitrogen Base without amino acids) containing 0.002% uracil). Saccharomyces cerevisiae YPH499-U, YPH499-UL, YPH499-ULH, YPH499-ULHA, and YPH500-U, YPH500-UL, YPH500-ULW, YPH500- are selected from the grown yeasts and are genome-integrated CALB arming yeasts. ULWA arming yeast was obtained

Table 2 Restriction enzymes used for cleavage in the selection marker of each plasmid

(2-B. Preparation of Haploid Saccharomyces-CALB Arming Yeast)
Saccharomyces cerevisiae YPH499-U, YPH499-UL, YPH499-ULH, YPH499-ULHA, and YPH500-U, YPH500-UL, YPH500-ULW, YPH500-ULWA arming yeasts created in 2-A above were added to SDC medium (2 % Glucose, 0.67% Yeast Nitrogen Base without amino acids, 2% casamino acid, applied amino acids and nucleic acids), and osmotically cultured at 30 ° C. for 6 days. Subsequently, it isolate | separated into the culture medium and the microbial cell by centrifugation, and let the obtained microbial cell be the haploid Saccharomyces-CALB arming yeast. The obtained haploid Saccharomyces-CALB arming yeast was confirmed for lipase activity at 30 ° C. using p-nitrophenyl butyrate (hereinafter abbreviated as PNPB) as a substrate.

(Creation of 2-C. Diploid Saccharomyces-CALB Arming Yeast)
The haploid arming yeasts YPH499-ULHA and YPH500-ULWA are inoculated into YPD medium (2% glucose, 2% peptone, 1% yeast extract), cultured at 30 ° C. for 1 hour, and then centrifuged to form a medium and cells. The bacterial cells obtained after separation were mixed, resuspended in YPD medium, and cultured at 30 ° C. overnight. This was diluted to an appropriate cell density, and then cultured using SD + K agar medium (2% glucose, 0.67% Yeast Nitrogen Base without amino acids, 0.002% L-lysine). The grown yeast was selected to obtain Saccharomyces cerevisiae YPH501-U2L2HWA2 arming yeast, which is a genome-introduced diploid CALB-arming yeast.

(2-D. Preparation of Diploid Saccharomyces-CALB Arming Yeast)
Saccharomyces cerevisiae YPH501-U2L2HWA2 arming yeast obtained in 2-C above is added to SDC + K medium (2% glucose, 0.67% Yeast Nitrogen Base without amino acids, 2% casamino acid, 0.002% L-lysine) Inoculated and osmotic cultured at 30 ° C. for 6 days. Subsequently, it isolate | separated into the culture medium and the microbial cell by centrifugation, and confirmed the lipase activity (ester decomposition activity) in 30 degreeC of the microbial cell which used PNPB as the substrate.

  The PNPB method was used for the measurement of ester decomposition activity. The cells were collected by centrifuging the culture solution, and the obtained cells were washed twice with distilled water. Subsequently, the cells were suspended in 20 mM phosphate buffer and preincubated at 30 ° C. for 5 minutes. On the other hand, PNPB was used as the substrate. PNPB was dissolved in a small amount of ethanol and then diluted appropriately with distilled water to prepare a substrate solution. The substrate solution was added to the pre-incubated cell suspension, stirred well, and allowed to react at 30 ° C. for 10 minutes while shaking. Subsequently, 5% trichloroacetic acid aqueous solution was added to stop the reaction, followed by centrifugation. The supernatant was collected, diluted with 200 mM phosphate buffer, and the absorbance at 400 nm was measured. Then, the amount of p-nitrophenol (PNP) produced was determined and used as ester decomposition activity. In defining the activity, the amount of enzyme that PNP produces at 1 μmol per minute was defined as 1 U.

It is a GC chart of dehydrated castor oil fatty acid DCO / FA used in the reaction. 10 is a GC chart of the product in Example 7. Since the molecular weight m / z of the peak observed at a holding time of 18 to 20 minutes is the molecular weight of HEMA added to the peak of the raw material (FIG. 1), it can be understood that it is the target product. It is a schematic diagram of plasmid pUC19-ProCALB CBS6678 preparation in a manufacture example. It is a schematic diagram of CALB surface layer expression cassette preparation in a manufacture example. It is a schematic diagram of CALB surface layer expression plasmid preparation in a manufacture example.

Claims (7)

In the presence of lipase (A) or arming yeast (B) presenting lipase on the cell surface, general formula (1)

(In the formula, R ₁ represents a hydrogen atom or a methyl group, X represents an oxygen atom or a nitrogen atom, and n represents an integer of 2 to 4.)
(Meth) acrylic acid derivatives represented by the general formula (2)

(In the formula, R ₂ represents a C 5-23 alkyl group or alkenyl group which may have a hydroxyl group or a carboxy group.)
A carboxylic acid represented by the general formula (3)

(In the formula, R ₁ is a hydrogen atom or a methyl group, X is an oxygen atom or a nitrogen atom, n is an integer of 2 to 4, and R ₂ is an alkyl group having 5 to 23 carbon atoms which may have a hydroxyl group or a carboxy group. Or represents an alkenyl group.)
The manufacturing method of the acrylate compound represented by these.   The lipase (A) is selected from the genus Aspergillus oryzae, the genus Achromobacter, the genus Bacillus, the genus Candida, the genus Chromobacter, the genus Fusarium, and Fusarium. 2. An acrylate obtained from a microorganism of the genus Hypozyma, Pseudomonas, Rhizomucor, Rhizopus, or Thermomyces, which is a genus of Thermomyces. Production method.   The arming yeast (B) presenting the lipase on the cell surface layer is a diploid arming yeast, and is characterized by cell fusion of a haploid a cell arming yeast and a haploid α cell arming yeast. The manufacturing method of the acrylate compound of Claim 1.   The arming yeast (B) presenting the lipase on the cell surface is a secretory signal sequence wherein the protein displayed on one or both of the haploid a cell arming yeast and haploid α cell arming yeast is the secretory signal sequence. The structural gene sequence of the protein, a sequence encoding a part of a cell surface localized protein, and a sequence encoding a GPI anchoring domain are expressed on the cell surface by DNA having a sequence in this order. The manufacturing method of the acrylate compound of this. The arming yeast (B) presenting the lipase on the cell surface is a secretory signal sequence wherein the protein displayed on one or both of the haploid a cell arming yeast and haploid α cell arming yeast is the secretory signal sequence. The method for producing an acrylate compound according to claim 3, which is expressed on the cell surface by DNA having the structural gene sequence of the protein and the sequence encoding the GPI anchoring domain in this order. The arming yeast (B) presenting the lipase on the cell surface is a secretory signal sequence wherein the protein displayed on one or both of the haploid a cell arming yeast and haploid α cell arming yeast is the secretory signal sequence. , A structural gene sequence of the protein, a DNA having a sequence encoding a sugar chain binding protein domain in this order, or a secretory signal sequence, a sequence encoding a sugar chain binding protein domain, a structural gene of the protein presented to the cell surface The method for producing an acrylate compound according to claim 3, wherein the acrylate compound is expressed on the cell surface by DNA having sequences in this order.   The method for producing an acrylate compound according to any one of claims 3 to 6, wherein the haploid a-cell arming yeast and the haploid α-cell arming yeast are Saccharomyces yeasts.

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